Centromere (CEN) DNA from yeast can be isolated, inserted into plasmids, and shown to exhibit the basic behavioral characteristics of intact centromeres in yeast. Centromere function in whole chromosomes can be investigated by substitution of the chromosomal CEN by mutant CEN DNA. We are using these approaches to investigate the molecular details of centromere function in mitotic and meiotic chromosme movement. It is now firmly established that the centromere regions of all yeast chromosomes are similar in general organization and have in common certain CEN DNA sequence elements. These include an 8 bp sequence, CDE I (PuTCACPuTG), a central A+T rich core segment of 78-86 bp (CDE II), and a 25 bp region, CDE III (TGT-T-TG--TTCCGAA-----AAA). We have constructed and analyzed a library of mutations in these conserved CEN sequences and have identified specific nucleotides in CDE III that are essential for chromosome stability during mitosis. Our results suggest that CDE III is a recognition site for a centromere DNA binding protein. Results from functional analyses of our CDE II replacement mutants have begun to delineate the base composition and structural organization required for function of that segment of CEN DNA. In the proposed research, we will extend the CEN mutant studies and use physical and genetic techniques to probe the three dimensional architecture of the yeast kinetochore. One immediate goal of this work is to determine which CEN sequence elements function in mitosis and which function in meiosis. We will also investigate the in vivo interactions among the conserved sequence elements by selecting for cisacting mutations in CDE I that suppress the functional defects in CDE III mutants. We will probe kinetochore architecture by using an in vivo footprinting strategy to identify contact points between the centromere DNA and the kinetochore proteins. Isolation of the genes encoding the centromere DNA binding proteins will be attempted using an in vivo genetic selection. The CEN-flanking DNA will be analyzed for transcription termination signals that act to prevent transcriptional inactivation of the centromere in vivo. The long range goal of this work is to understand the details of centromere function and structure in yeast. The single microtubule attachment site in yeast may, in fact, be a model unit repeat structure for the multiple attachment sites of the more complex kinetochores typical of other eucaryotic chromosomes.